1. Field of the Invention
The present invention relates to methods for producing proteins secreted out of plant cells.
2. Description of the Related Art
When a heterologous protein gene is introduced into a plant host cell and is highly expressed, secreting the expressed heterologous protein into the extracellular apoplast to accumulate therein may be envisioned as a measure to reduce a load on the host cell.
Methods for secreting a heterologous protein into an extracellular apoplast may include a method of expressing the heterologous protein in the form of a fusion protein in which a signal sequence that directs endoplasmic reticulum (ER) transport is linked to the amino terminus of the heterologous protein. The signal sequence allows the linked protein to penetrate into the endoplasmic reticulum lumen, and the signal sequence is cleaved from the heterologous protein by a signal peptidase in the ER membrane to release the heterologous protein into the ER lumen. The heterologous protein is folded in the ER lumen, and then secreted into the extracellular apoplast by the intracellular transport system.
Glycinin is a soybean seed storage protein, and after it is translated in the form of a proglycinin precursor having a signal sequence for ER transport, the signal sequence is cleaved from the precursor in the ER membrane to release a proglycinin. Proglycinin forms a trimer in the EA and is transported into a protein storage vacuole, and then is processed into a mature glycinin to form a hexamer. Maruyama et al. (The Plant Cell, 2006, vol. 18, p. 1253-1273) reported that in an experiment of transiently expressing a green fluorescent protein (hereinafter, sometimes referred to as GFP) in a soybean immature seed, only when a glycinin signal sequence for ER transport that was flanked on its carboxy terminus by 9-amino acids sequence derived from a proglycinin amino-terminal region was fused to the amino terminus of a GFP to express, the GFP was secreted into the extracellular apoplast. Kawagoe et al. (The Plant Cell, 2005, vol. 17, p. 1141-1153) reported that in an experiment of expressing a GFP in a recombinant rice immature seed, when a glycinin signal sequence for ER transport that was flanked on its carboxy terminus by 2-amino acids sequence derived from a proglycinin amino-terminal region and a few other amino acids was fused to the amino terminus of a GFP to express, the GFP was not secreted into the extracellular apoplast.
In production of useful proteins, to maintain original useful properties such as enzyme activities or physiological functions of an intended heterologous protein, it is preferred that heterologous proteins to be produced have no additional heterologous sequences or short ones, if any.